The ability of some of the strains of Escherichia coli to attach to HEp-2 cells in tissue culture has been correlated with their capacity to cause diarrheal illness. Three distinct patterns of adherence to HEp-2 cells have been described by investigators at the Center for Vaccine Development of the University of Maryland School of Medicine:
(1) localized adherence, PA1 (2) diffuse adherence, and PA1 (3) aggregative adherence. PA1 (1) It requires tissue culture facilities. PA1 (2) It takes four hours to run. PA1 (3) It is expensive. PA1 It is not amenable to testing large numbers of the population and PA1 (5) It is subject to observer error and therefore requires experienced personnel. PA1 HEp-2 CELLS: These are tissue culture cells utilized in an adherence assay for identification of different types of E. coli bacteria types. PA1 EAF PLASMID: This is a plasmid which is present in E. coli strains of bacteria exhibiting localized adherence. ENTEROPATHOGENIC E. COLI (EPEC): This is a category of diarrheagenic E. coli bacteria exhibiting a localized adherence pattern when tested with HEp-2 cells. PA1 ENTEROAGGREGATIVE E. COLI: These strains of E. coli show a correlation with diarrhea. PA1 DIFFUSE ADHERENCE PATTERN: This is exhibited by some E. coli strains. It is not agreed as to whether these bacteria are associated with diarrhea. PA1 LOCALIZED ADHERENCE PATTERN: This is exhibited by enteropathogenic E. coli (EPEC) in the HEp-2 assay. PA1 AGGREGATIVE PATTERN: This pattern is exhibited by a enteroaggregative E. coli in the presence of HEp-2 cells in culture. PA1 HOMOLOGOUS DNA: Is a DNA fragment which is from a microorganism or from a closely related microorganism. PA1 NON-HOMOLOGOUS DNA: Is a DNA fragment which in general does not hybridize with a DNA segment. PA1 VECTOR: This is a DNA segment which can replicate itself in a host by utilizing the host's machinery. Vectors are useful as cloning tools for replicating other DNA segments fused thereto. PA1 FUNCTIONAL EQUIVALENT DNA SEGMENTS: These are DNA sequences which may have a certain percentage of variation from one another while still preserving a defined characteristic, e.g., translation into a polypeptide, hybridization to a specific DNA segment, and the like. PA1 ACTIVE DNA FRAGMENTS: These are portions of a DNA segment of a lesser number of bases which still preserve a certain characteristic possessed by the complete segment, e.g., hybridization to a determined DNA. PA1 NUCLEOTIDE: Is the basic molecule of DNA and is made up of a base, a sugar and a phosphate group. PA1 DEOXYRIBONUCLEIC ACID (DNA): This is a double-stranded chain of deoxyribonucleotides. Each nucleotide in one strand has a corresponding or complementary nucleotide at the same position of the opposite strand. Some DNAs encode proteins, with each trinucleotide encoding a single amino acid. PA1 RIBONUCLEIC ACID (RNA): This is a chain of ribonucleotides. PA1 OLIGONUCLEOTIDE: This is a portion of a DNA fragment consisting of a few to several hundred nucleotides. PA1 NUCLEOTIDE SEQUENCE: This is a linear chain of nucleotides along a DNA strand in the exact order of their appearance in a DNA fragment. PA1 RESTRICTION ENDONUCLEASES: These are enzymes that cut or restrict the DNA molecule into oligonucleotide fragments. Each enzyme cuts at specific and constant sites PA1 LIGATION: This is a process whereby an enzyme or a ligase splices or ligates together two or more DNA fragments by means of a chemical bond. PA1 VECTOR: This is a DNA sequence or molecule that can autonomously replicate in a host. Examples are plasmids and viruses. PA1 GENETIC ENGINEERING/RECOMBINANT DNA: Technology relying on the manipulation of a DNA fragment from one source that is transferred to and can replicate in a host. Typically, a DNA fragment of choice is restricted from a larger DNA molecule, ligated to an appropriately restricted vector, and transformed into a new host cell. PA1 CLONE: This term refers to a population of cells whose members carry one or more copies of a specific recombinant DNA fragment. PA1 PLASMID DNA: This is a segment of DNA which is carried by the host not as part of its genome but as separate therefrom. PA1 GENE LIBRARY OR GENE BANK: This is a collection of clones representing all or a considerable portion of the DNA contained in an organism. PA1 GENE: This term describes a DNA sequence that contains information for construction of a polypeptide or protein. It includes 5' and 3' ends thereof. PA1 SUBCLONING: This term refers to a process of removing extraneous DNA from a recombinant DNA fragment. PA1 HYBRIDIZATION: By this is meant the formation of a double stranded DNA between two single strands of DNA or one RNA and one DNA strand derived from the same or different sources PA1 PROBE: Is a DNA fragment labeled for purposes of monitoring hybridization reactions. PA1 POLYMERASE CHAIN REACTION (PCR): This term refers to a process of enzymatic amplification of a portion of a DNA fragment. The portion that is amplified is selected by the choice of primers that flank each side of the DNA to be amplified. PA1 PRIMER: Is an oligonucleotide that will allow the polymerase chain reaction to proceed, if its sequence is identical, or nearly so, to that of the two regions that flank the region of the DNA to be amplified. PA1 EXPRESSION VECTOR: Is a cloning vector which contains a strong promoter. Such a vector can allow the efficient transcription of a gene inserted into it, and is useful when the objective of cloning is to obtain high levels of a particular gene product. PA1 PROMOTER: Can be inserted into a DNA strand. A promoter is a nucleotide sequence which is recognized and bound by a DNA-dependent RNA polymerase during the initiation of transcription. It is usually in the 5' region of a gene and is located proximal to the start codon. PA1 TRANSCRIPTION: Is the synthesis of an RNA strand from a complementary DNA strand in a process in which ribonucleotide 5'-triphosphates bases pair sequentially with nucleotides in a template strand (the DNA strand) and are polymerized in the 5' to 3, direction by an RNA polymerase. PA1 TERMINATOR: Is a segment of DNA that specifies the termination of a transcriptional signal. The secondary structure in the transcript itself appears to be important in effecting termination. In bacteria terminators vary in efficiency and in mechanism of action. PA1 TRANSFORMATION: Is a procedure by which exogenous DNA is taken up by a recipient or host cell. The exogenous DNA may be incorporated into the chromosome or a plasmid of the host by homologous recombination, or it may be converted into an autonomous replicon. PA1 REPLICON: Is any DNA fragment or molecule which possesses a replication origin and which is therefore potentially capable of being replicated in a suitable cell. PA1 STRUCTURAL GENE: Is a DNA sequence that may be transcribed into a messenger RNA, which may then be translated into a specific polypeptide having a characteristic sequence of amino acids. PA1 HETEROLOGOUS DNA: Is a DNA fragment which is foreign to, or originates from a different organism than, the host, chemically synthesized gene or fragments thereof. A heterologous gene or fragments thereof code for polypeptides ordinarily not produced by the organism susceptible to transformation by the expression vector or by other structural genes cloned therein. PA1 OPERABLY LINKED: Is the in-frame splicing of a DNA segment such that the promoter controls the initiation of the expression of the polypeptide encoded by the structural gene. PA1 EXPRESSION: Is a process by which a structural gene produces a polypeptide. The process involves transcription of the gene into a messenger RNA (mRNA) and translation thereof into a polypeptide chain. PA1 VEHICLE: Is the organism in which a recombinant DNA molecule is replicated during cloning experiments. PA1 CLONING (OF DNA): Is an in vitro procedure in which a particular DNA sequence, e.g., a gene or fragments thereof, is reproduced in large amounts by inserting or splicing it into a suitable replicon (the vector or cloning vector), introducing the resulting recombinant or hybrid molecule into a host cell where it can replicate, and finally growing the cells in culture. The DNA to be cloned, e.g., the donor, exogenous, or foreign DNA, may be obtained by extracting DNA from an organism and subjecting it to mechanical shearing or endonuclease enzyme action. The vector is typically either a plasmid or a viral genome. PA1 INDUCIBLE: It is said that a gene or gene-product is inducible if its transcription or synthesis is enhanced or increased by exposure of the cells to an effector. An inducible heat-shock promoter is induced by the presence of high temperature which effects significant changes in the regulatory factors governing increased transcription rates. PA1 GRAM NEGATIVE: Cells which lose the primary violet or blue color during decolorization in Gram's staining method. The method, developed by Hans Gram, a Danish physician, in 1884, gives a simple and convenient distinction between groups of bacteria. The staining reaction reflects differences in cell wall composition, but the mechanism is not clear. PA1 GRAM POSITIVE: Cells which retain the primary violet or blue stain in Gram's method. PA1 IN VITRO: In latin it means "in glass", that is to say not in a living animal or person. PA1 IN VIVO: In latin it means in a living animal or person. PA1 PATHOGENIC: This term refers to the production of disease or pathological changes. PA1 IN-FRAME: It is said that two oligonucleotide open reading frames maintain their single contiguous open reading frames after ligation if the ligated product contains the same sequence of amino acids as that of the two separate oligonucleotide-coded products. PA1 FUSION: Is a process of in-frame ligation. PA1 OPERON: Is a fusion of two (bicistronic) or more genes with the same transcriptional unit. Their coordinate transcriptional regulation is implied of all genes contained within the same operon. PA1 PROCARYOTIC: This term encompasses technology related to bacteria (without nuclei) PA1 EUCARYOTIC: This term relates to organisms more advanced than bacteria (with nuclei).
The ability of E. coli strains to manifest localized adherence is dependent on the presence of a plasmid, the EPEC Adherence Factor (EAF) plasmid. The property of localized adherence is characteristic of a category of diarrheagenic E. coli referred to a enteropathogenic E. coli (EPEC). In several recent studies it has been shown that E. coli strains that manifest localized adherence are found significantly more often in cases of infant diarrhea than in matched controls
The genes responsible for the diffuse pattern of adherence to HEp-2 cells appear to be located in the chromosome. While E. coli that manifest the diffuse adherence pattern clearly represent a distinct category of E. coli, there is much disagreement as to whether they are associated with diarrhea. In some studies strains exhibiting diffuse adherence have been found significantly more often in test cases than in controls However, in several other studies this has not been found to be so the case.
E. coli that show the aggregative pattern in the HEp-2 cell assay (enteroaggregative E. coli) have been incriminated in two separate studies as diarrheal pathogens.
In 1987, one of the present inventors at the University of Maryland coined the term enteroadherent aggregative E. coli to refer to the bacterium exhibiting aggregative behavior in the HEp-2 cell assay. The group characterized the E. coli strains and considered them a putative new category of diarrhoegenic E. coli (Vial, P. A., et al., supra). In subsequent publications the term was shortened from enteroadherent aggregative E. coli to enteroaggregative E. coli. These enteroaggregative E. coli are distinct from the other categories of diarrheagenic E. coli in that they have the ability to attach to HEp-2 cells in tissue culture with a particular, "aggregative", pattern. This ability has been correlated to their capacity to cause diarrheal illness.
There are several features of enteroaggregative E. coli bacteria that show them to be distinct from other categories of diarrheagenic E. coli (Vial, P. A., Robins-Browne, R., Lior, H., Prado, V., Kaper, J. B., Elsayed, A., Levine, M. M., "Characterization of enteroadherent-aggregative Escherichia coli, a putative agent of diarrheal disease", J. Infect. Dis. 158:70-79 (1988), the entire content of which is incorporated herein by reference). Enteroaggregative E. coli possess a virulence plasmid that is distinct from the virulence plasmids of other diarrheagenic E. coli and fall into O:H serotypes that are distinct from other categories of diarrheagenic E. coli. Moreover, in animal models, enteroaggregative E. coli cause histopathological changes that are distinct from those caused by other categories of diarrheagenic E. coli.
In a study conducted in Santiago, Chile, enteroaggregative E. coli were isolated significantly more often from symptomatic cases than from matched controls. In another study carried out in rural India enteroaggregative E. coli were found significantly more often in cases of persistent diarrhea (&gt;14 days duration) (30%) than in cases of acute diarrhea (12.8%) or controls (9.9%). However, in a study undertaken in Brazil, enteroaggregative E. coli were isolated with similar frequency from cases of acute diarrhea and controls.
In recent years, DNA probes have been developed for identifying many of the categories of diarrheagenic E. coli common in epidemiologic studies. Sensitive, specific and reliable DNA probes already exist for dentifying enteropathogenic, enterotoxigenic, enteroinvasive and enterohemorrhagic E. coli as well as for E. coli exhibiting diffused and localized adherence to HEp-2 cells (Nataro, J. P., Baldini, M. M., Kaper, J. B., Black, R. E., Bravo, N., Levine, M. M., "Detection of an adherence factor of enteropathogenic Escherichia coli with a DNA probe", J. Infect. Dis., 152:560-565 (1985); Levine, M. M. et al, "Use of DNA probes and HEp-2 cell adherence assay to detect diarrheagenic E. coli", J. Infect. Dis., 158:224-228 (1988); Echeverria, P. et al, "Case-control study of endemic diarrheal disease in Thai children", J. Infect. Dis. 159:543-548 (1989); Lanata, C. F. et al, "Sensitivity and specificity of DNA probes with the stool blot technique for detection of Escherichia coli enterotoxin", J. Infect. Dis. 152:1087-1090 ( 1985); Mosley, S. et al, "Identification of enterotoxigenic Escherichia coli by colony hybridization using three enterotoxin gene probes", J. Infect. Dis. 145:863-869 (1982); Wood, P. K. et al, "Comparison of DNA probes with the Sereny test for identification of invasive Shiqella and Escherichia coli strains", J. Clin. Microbiol. 24:498-500 (1986); Levine, M. M. et al, "A DNA probe to identify enterohemorrhagic Escherichia coli of 0157:H7 and other serotype that cause hemorrhagic colitis and hemolytic uremic syndrome", J. Infect. Dis. 156:175-182 (1987), the entire contents of which are incorporated herein by reference).
Enteroaggregative E. coli (EAggEC) bacteria, however, do not hybridize with DNA probes that identify other categories of diarrheagenic E. coli. Presently, EAggEC bacteria can only be identified by means of a tissue culture assay and this cell assay (HEp-2) has many disadvantages among which are the following.
These drawbacks render the HEp-2 tissue culture assay ill suited for use in third world countries.
A previous search for a probe for enteroaggregative E. coli (EAggEC) produced a DNA fragment obtained from a plasmid from the 042 enteroaggregative E. coli strain (Vial, P.A. et al, "Characterization of EAggEC, a putative agent of diarrheal disease", J. Inf. Diseases 158(1): 70 (1988), the entire content of which is incorporated herein by reference). That fragment, however, was capable of hybridizing to no more than 20 out of 41 strains of EAggEC different from the 042 strain. This overall low sensitivity (49%) for enteroaggregative E. coli bacteria renders the 042 EAggEC fragment a poor tool for the systematic identification of enteroaggregative E. coli bacteria.
Accordingly, since the HEp-2 cell assay differentiating enteroaggregative E. coli on the basis of adherence is cumbersome, time-consuming, labor-intensive and subject to considerable error there remains a need for an easier, more reliable method which is adaptable for use in developing countries.